The present invention is directed to processes of isolating purified compositions with or without chromium containing compounds, from a variety of natural sources including, but not limited to, a yeast strain S. carlsbergensis, or S. cerevisiae, or any commercial source of yeast extract; or from the Saltbush, Atriplex halimus, growing in the Negev Desert near the Dead Sea and other arid areas in the middle east. The invention also comprises production of synthetic sources of chromium, including, but not limited to chromium gluconate, chromium sulfate, chromium-cysteine, chromium-N-acetyl cysteine, chromium-glutathione, chromium acetate, chromium citrate, chromium ascorbate or chromium tararate. The invention is also directed to formulations with or without chromium containing natural compounds or the synthetic chromium complexes, which specifically regulate glucose tolerance, glucose and lipid metabolism, insulin action, and metabolic activities in mammals who may be at an enhanced risk for or having a diabetic condition or cardiovascular diseases. In the practice of the method of treating diabetes, compositions containing the chromium complexes or the natural compositions with or without chromium containing compounds are used to regulate the diabetes related metabolic disregulation without general cytotoxic effects.
Chromium (Cr) has been known as an essential trace element in animal and human nutrition. Cr deficiency may result in various symptoms including increased concentrations of circulating insulin, elevated blood glucose and cholesterol, decreased insulin receptor number, elevated triglyceride and free fatty acid levels and reduced high-density lipoprotein (HDL) cholesterol levels. These metabolic abnormalities, in general, are associated with risk for and/or incidence of diabetes. Moreover, chromium levels in most organs decline with age and in diabetics the chromium levels are even lower-than in other healthy adults. Anderson, R. A., Clin. Physiol. Biochem. 4: 31-41 (1986).
Malnutrition associated with Cr deficiency brings an impaired glucose intolerance. However, inorganic Cr compounds are poorly absorbed by the gut, whereas organic Cr components are well absorbed in the body. Glucose Tolerance Factor (GTF) is an organic complex of chromium present in several natural sources, the richest among them is Brewer""s yeast. GTF was found to improve glucose tolerance in 50% of elderly patients with impaired glucose tolerance after two months of treatment. Despite these effects, the structure of GTF has not been identified as of yet Several naturally occurring organic Cr compounds have been proposed to serve as GTF, for example, a partially purified cationic Cr compound extracted from yeast, soluble in water and has an absorption spectrum at 260 nm, or the low-molecular-weight Cr-binding substance isolated from mouse or rabbit liver or bovine colostrum which has anionic properties. These organic Cr compounds have heretofore been used as crude extracts and the properties of their individual components remain uncharacterized. Evans, G. W. et al., Biochem. Biophys. Res. Commun. 50:718-722 (1973).
Hwang et al (U.S. Pat. No. 4,985,439) attempted to purify the Glucose Tolerance Factor from autolyzed brewer"" yeast and claimed that the active fraction is a quinoline derivative. King, S. (U.S. Pat. No. 5,108,610) also tried to isolate and purify a material possessing Glucose Tolerance Factor activity from eukaryotic cell mass, and claimed that the active material is dithiochrome. In addition, some patents describe a number of synthetic Cr compounds, (Furman C. S. et al, U.S. Pat. No. 5,266,560; Jensen N. L., U.S. Pat. No. 5,194,615; Wong Y., U.S. Pat. No. 5,536,863; Evans G. W., U.S. Pat. No. 5,087,624 and U.S. Pat. No. 4,315,927; Hwang D. et al, U.S. Pat. No. 4,985,439). Some patents describe chromium picolinate as a hypoglycemic chromium compound (U.S. Pat. No. 5,087,623; U.S. Pat. No. 5,087,624; and U.S. Pat. No. 5,175,156). The present invention is directed to development of improved processes to isolate natural compositions, with or without active and stable organic Cr compounds, as well as having hypoglycemic and/or hypolipidemic activity, from natural sources including, but not limited to, Brewer""s yeast and Saltbush plant.
The present invention relates to compositions having hypoglycemic and/or hypolipidemic activity with or without natural or synthetic chromium compounds for application to individuals at risk for or suffering from diabetes, CVD, and cell proliferative diseases by regulation of metabolic abnormalities and/or inhibition of metabolic abnormalities within the target cells. In the practice of the invention, the natural or synthetic chromium compounds can be applied to supplement traditional pharmaceutical, hormonal and/or nutritional therapies for diabetes.
Diabetes mellitus is a disease of metabolic disregulation, notably of glucose metabolism, and long-term vascular and neurologic complications. Diabetes has several clinical forms, the two major forms being insulin-dependent diabetes mellitus I (IDDM) and the non-insulin-dependent diabetes mellitus II (IDDM). IDDM is rare, affecting one in 250 persons in the United States, where approximately 10,000 to 15,000 new cases are reported each year. Data suggest that the incidence of IDDM is increasing in Europe, where the highest prevalence is found in northern Europe, for example, more than one in every 150 Finns develop IDDM by 15 years of age. LaPorte, R. et al., in Diabetes in America, 2nd ed. Ed M. Harris, National institutes of Health, Bethesda, Md. NIH Publication No 95-1498, 1995.
NIDDM is common, with an overall prevalence of 6.6 percent in the United States. NIDDM has become one of the most frequent chronic diseases in most industrialized nations and the projected prevalence for the next decade is 10 percent. 600, 000 new cases are reported each year and one half of the NIDDM population are unaware of their disorder. The increase in the prevalence of NIDDM in the United States is commonly attributed to an aging population that is also increasingly obese and sedentary. The prevalence of NIDDM among persons older than 65 years exceeds 18 percent, and compared with normal-weight individuals, obese people with a body mass index greater than 30, are at 10 to 20 times greater risk for NIDDM. Although genetic and immunologic markers for IDDM have been identified, they are not enough nor sensitive enough to be used to define IDDM or distinguish IDDM and NIDDM. Harris, M. I., et al., Diabetes 36: 523 (1987); Bennett, P. H., et al., in International Textbook of Diabetes, ed Alberti KGMM, et al., John Wiley and Sons Ltd UK 1992, p148.
In accordance with the invention, improved processes of isolating from natural sources, compositions having hypoglycemic and/or hypolipidemic activity, with or without chromium containing natural extracted compounds are provided for use in individuals and animals at risk for or suffering from diabetes CVD and other cell proliferative diseases. The natural sources used include, but are not limited to the yeast strains S carisbergensis and S cerevisiae, and the Saltbush Atrirplex halimus growing in the Negev Desert near the Dead Sea and other arid areas in the middle east.
The present invention also provides processes to produce synthetic sources of chromium complexes including, but not limited to, chromium gluconate, chromium sulfate, chromium-cysteine, chromium-N-acetyl cysteine, chromium-glutathione, chromium acetate, chromium citrate, chromium ascorbate or chromium tartate.
The present invention also provides methods for the synthesis of chromium complexes, which are pure and can be prepared in large quantities without possibility of contamination.
The present invention provides formulations having hypoglycemic and/or hypolipidemic activity, with or without chromium containing natural and synthetic compositions exhibiting GTF, activity, for application in a variety of diseases or conditions including, glucose intolerance, hyperlipidemia, hypercholesterolemia, obesity, vascular and fibrotic proliferative diseases, skin lesions, diabetic neuropathy or to regulate abnormal metabolic processes associated with diabetes, or CVD.
The present invention provides formulations having hypoglycemic and/or hypolipidemic activity, with or without chromium containing natural and synthetic complexes which can be applied in combination with an effective amount of one or more additional antioxidants including vitamin C, vitamin E, reduced glutathione, manganese, beta-carotene, ergothioneine, zinc, selenium, cysteine, N-acetyl cysteine, methionine or 2-mercaptoethanol.
According to an additional aspect of the present invention, there is provided a method to regulate glucose metabolism, insulin activity, lipid metabolism and lipoprotein lipase activities by applying formulations having an effective amount of hypoglycemic and/or hypolipidemic activity, with or without chromium coning natural and synthetic chromium compositions.
According to yet another aspect of the invention, there is provided a method to regulate glucose metabolism, insulin activity, lipid metabolism and lipoprotein lipase activities by applying formulations having hypoglycemic and/or hypolipidemic activity, with or without chromium containing natural and synthetic chromium compositions in combination with an effective amount of one or more antioxidants including vitamin C, vitamin E, reduced glutathione, manganese, beta-carotene, ergothioneine, zinc, selenium, cysteine, N-acetyl cysteine, methionine or 2-mercaptoethanol.
The present invention also provides a method to regulate glucose metabolism, insulin activity, lipid metabolism and lipoprotein lipase activities, including application of formulations of chromium containing natural and synthetic chromium compositions m combination with a conventional therapeutic regimen including hormonal therapy or one or more pharmaceutical agents.
The present invention is based on the discovery of improved processes to produce more purified compositions having hypoglycemic and/or hypolipidemic activity, with or without chromium containing natural extracted compounds.
The present invention is based on the discovery of improved processes to produce synthetic compositions of chromium compounds.
The present invention is also based on the discovery of improved formulations of chromium containing natural and synthetic compositions which exhibit a greater potency in regulating metabolic abnormalities associated with diabetes and obesity.
It is also the object of the present invention to provide formulations having hypoglycemic and/or hypolipidemic activity, with or without chromium containing natural and synthetic compositions which are effective when used alone or in combination with antioxidants or conventional therapies.
It is an object of the present invention to provide methods for the synthesis of chromium complexes which exhibit GTF activity.
It is also an object of the present invention to provide methods for the synthesis of chromium complexes which are pure and can be prepared in large quantities without possibility of contamination.
It is another object of the present invention to provide methods for prevention and treatment of diabetes-related abnormalities in glucose and lipid metabolism, or insulin and lipoprotein lipase activities.
Still other objects and advantages of the invention will in part be obvious and will in part be apparent from the specification.
The invention accordingly comprises the several steps and the relation of one or more of such steps with respect to each of the others, and the product embodying properties, which are adapted to effect such steps and methods, all as exemplified in the following detailed disclosure, and the scope of the invention will be indicated in the claims.
For a fuller understanding of the invention, reference is had to the following description taken in connection with the accompanying drawings, in which:
FIG. 1 is a diagram illustrating the individual steps in the process by which the chromium containing natural compositions are extracted and purified from yeast strain or yeast extract. These steps include: a) mixing of crude yeast preparation with a chloroform/methanol/water solvent, b) separation of the aqueous phase, c) mixing of the aqueous fraction with ethanol, and d) isolating the active sediment fraction, e) mixing the sediment with methanol f) ultra-filtration of methanol eluate g) collection of the active fraction below 1000 dalton.
FIG. 2 is a diagram illustrating individual steps in the process by which the chromium containing natural compositions are extracted and purified from the Saltbush. These steps include: a) preparation of a crude mixture of Saltbush, b) mixing of the crude preparation with a chloroform/methanol/water solvent, c) separation of the aqueous phase, d) mixing of the aqueous phase with ethanol, e) separation of the active component in the eluate, f) mixing of the active component with methanol, and separation of the eluate containing the active fraction.
FIG. 3 is an elution profile of 100% methanol elute from Saltbush on preparative HPLC C18 column. The mobile phase used: H2O +1% ammonium acetate; 100% H2O; a gradient between 100% water to 100% acetonitrile. Several peaks were observed at 250 nm. The activity of the isolated fractions was measured in yeast fermentation assay. Fraction 1 was found to be most active. Fractions 6-7 showed also high activity whereas all the other isolated fractions were inactive.
FIG. 4 is a Mass Spectra (negative ion mode) of Fraction 1, from Saltbush extract (Ah) eluted from HPLC, by electrospray mass spectrometry (EP-Sciex, EPI 2000).
FIG. 5 is a Mass Spectra (negative ion mode) of Fractions 6-7, from Saltbush extract (Ah) eluted from HPLC, by electrospray mass spectrometry (EP-Sciex, EPI 2000).
FIG. 6 is a diagram illustrating carbon dioxide production in yeast cells in chromium depleted medium and after addition of active fractions of natural compositions extracted and purified from yeast.
FIG. 7 is a diagram illustrating carbon dioxide production in yeast cells in chromium depleted medium and after addition of active fractions of natural compositions extracted and purified from Saltbush.
FIG. 8 is a diagram illustrating carbon dioxide production in yeast cells in chromium depleted medium and after addition of different concentrations of chromium-ascorbate.
FIG. 9 is a diagram illustrating relative enhancement in fermentation rate for yeast cells in chromium depleted medium, and after addition of different concentrations of chromium ascorbate.
FIG. 10 is a diagram ill carbon dioxide production in yeast cells in chromium depleted medium and after addition of different concentrations of chromium glutathione.
FIG. 11 is a diagram illustrating relative enhancement in fermentation rate for yeast cells in chromium depleted medium, and after addition of different concentrations chromium glutathione.
FIG. 12 is a diagram illustrating carbon dioxide production in yeast cells in chromium depleted medium and after addition of different concentrations of chromium N-acetyl cysteine.
FIG. 13 is a diagram illustrating relative enhancement in fermentation rate for yeast cells in chromium depleted medium, and after addition of different concentrations of chromium N-acetyl cysteine.
FIG. 14 describes oral glucose tolerance test in diabetic rats which were either untreated or which were given an oral single dose of 140 ng Cr/rat of chromium containing natural compositions extracted from yeast. Diabetes type I was induced in rats with streptozotocin.
FIG. 15 describes glucose load in diabetic rats which were either untreated or which were given an oral single dose of 0.25, 0.5 or 1 g/rat of natural active compositions extracted from Saltbush. Diabetes type I was induced in rats with streptozotocin.
FIG. 16 describes glucose tolerance in diabetic spiny mice (Acomys russatus) suffering from diabetes type II which were either untreated or given a single oral dose of chromium containing natural composition extracted from yeast (200 ng Cr/animal).
FIG. 17 describes glucose tolerance in diabetic Sand rat (Psammomys Obesus) suffering from diabetes type II, which were either untreated or which were given a single oral dose of chromium containing natural composition extracted from Saltbush.
FIG. 18 describes the effect of chromium containing natural composition extracted from yeast on the activity of marginal levels of insulin in diabetes type I in rats.
FIG. 19 describes blood glucose levels in diabetic rats which were either untreated or which given an oral single dose of chromium containing natural composition extracted from yeast. Diabetes was induced by steroid treatment for several days.